A polyketide is a group of compounds containing a number of natural substances which vary in their structures and functions. Polyketides are known to include compounds having a variety of bioactivities such as antibacterial agents, antimyotic agents, antiparasitic agents, anti-insect agents, antitumor agents, and immunosuppressant agents, and aromatic compounds which are produced by bacteria, fungi and plants.
The above-mentioned various polyketide compounds are synthesized by the same biosynthetic mechanism which is very similar to the biosynthesis of fatty acids. That is, a polyketide compound is biosynthesized by the steps of continuous condensation of lower fatty acids including acetic acids and propionic acids, and subsequent reactions such as reduction of ketone, dehydration and enoyl reduction of each carbonyl group at β position of the extended acyl group which is similar to fatty acid synthesis. These various repetitive synthetic processes of many polyketide compounds are carried out a macromolecule, multifunctional enzyme complex, which has specific active sites (domains) required for each catalytic activity. A general reaction manner of polyketide biosynthesis is outlined, for example in Ann. Rev. Gen., 24, 37 (1990), and Ann. Rev. Microbiol., 47, 875 (1993).
It has been shown that a DNA sequence encoding polyketide synthase usually encodes all the required activities for the synthesis of a polyketide backbone. The DNA sequence encoding polyketide synthase is composed of modules, that is, repeating units involving condensation steps and modification steps following condensation. Each catalytic activity is involved in specificity to a specific carboxylic acid component of each condensation step, or in a different site which specifies a modification function following a specific condensation step to be achieved. For example, International Publication WO93/13663 describes the constitution of a gene encoding polyketide synthase of Saccharopolyspora erythaea. This gene consists of 6 modules, each of which is responsible for one condensation step. That is, a correct sequence of acyl side chain elongation and modification of an elongating chain are determined by genetic information present in each module.
Regarding the biosynthetic mechanism of avermectin aglycon, it has been reported that like other polyketide compounds, synthesis units of avermectin aglycon are lower fatty acids, such as acetic acid and propionic acid as its components [J. Antibiot., 39, 541-549 (1986)], and as in Saccharopolyspora erythaea, polyketide synthase consisting of modules is present in avermectin-producing bacteria [Gene, 115, 119-125 (1992), Ann. New York Acad. of Sci., 721, 123-132 (1994)].
Japanese Published Unexamined Patent Application No. 15391/91 describes a DNA fragment involved in avermectin biosynthesis, but shows no nucleotide sequence of the DNA fragment. This publication merely assumes the presence of polyketide synthase, which is involved in the synthesis of avermectin aglycon and the presence of partial modules. Therefore, the entire structure of polyketide synthase of avermectin cannot be predicted.
Similarly, MacNeil et al have reported a domain structure of partial modules [Ann. New York Acad. of Sci., 721, 123-132 (1994)]. However, they have not revealed the nucleotide sequence that should be evidence for polyketide synthase of avermectin.
Alteration of polyketide synthase would be a very useful breeding technique upon breeding of bacterial strains which can be used for a novel process for producing a novel avermectin useful as veterinary drugs and agricultural chemicals, and can produce a more effective avermectin derivative. Steps required to carry out such alteration include determination of the entire nucleotide sequence of a gene encoding polyketide synthase, accurate determination of a domain structure of each module based on the sequence, and introduction of a desired mutation. However, as described above, it was very difficult to carry out such improved-breeding, since the polyketide synthase gene of avermectin aglycon had not been specified and the nucleotide sequence of the gene was unknown.
The present inventors have studied approaches for producing a component different from that produced by the wild type strain by engineering DNA involved in polyketide synthesis with various methods. To apply this methodology, first we had to isolate a DNA molecule involved in the biosynthesis of a polyketide compound.
Hence, an object of the present invention is to provide a DNA encoding a multifunctional enzyme involved in biosynthesis of avermectin aglycon, and a process for producing avermectin aglycon, altered avermectin aglycon, avermectin, and altered avermectin using the DNA.